Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
  • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
  • C07K7/62—Polymyxins; Related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to novel polymyxin compounds, pharmaceutical compositions comprising the compounds, and the use of the compounds and the pharmaceutical compositions for medical treatment, for example treatment of microbial infections, particularly infections by Gram-negative bacteria.
• WO 2013/072695 and WO 2014/188178 describe polymyxin derivatives in which the N-terminal fatty acyl moiety and adjacent diaminobutyric acid moiety of Polymyxin B or Colistin are replaced by a terminal group having an amino substituent. Such derivatives have good antibacterial activity whilst having a reduced cytotoxicity.
• new polymyxin derivatives must at least match the activity of these known polymyxins whilst having significantly lower renal toxicity in vivo.
• the compound of formula (I) may also be represented thus:
• a compound of formula (I), or a pharmaceutical composition comprising a compound of formula (I), for use in a method of treating a microbial infection.
• the present invention also provides methods for the preparation of compounds of formula (I) as well as intermediate compounds for use in the preparation of the compounds of formula (I).
• the present invention provides compounds of formula (I), including the compounds of formula (II) and (III) as described in further detail below, for use in medical treatment, optionally together with a second active agent.
• the compounds of formula (I), (II) and (III) have a polymyxin core, which is a nonapeptide core, and a group —X—R 15 at the N terminal of the polymyxin core.
• the group —R 15 is a substituted ⁇ -aminopropyl group.
• the ⁇ -aminopropyl group is substituted at the ⁇ -position relative to the —X— moiety with an optionally substituted aryl or aralkyl group.
• a Dap residue diaminopropionic acid
• L-Dap L-Dab residue
• L-diaminobutyric acid L-diaminobutyric acid
• amino acid residues at positions 6 and 7 of the polymyxin compound may correspond to those amino acid residues present in Polymyxin B and Colistin.
• the amino acid residues at positions 6 and/or 7 may be substituted with different amino acid residues to those present within Polymyxin B and Colistin.
• new polymyxin derivatives must at least match the antibacterial activity of those known polymyxin compounds whilst having significantly lower renal toxicity in vivo.
• cytotoxicity refers to measured IC 50 relative to that recorded for Polymyxin B against a HK-2 cell line.
• the drug level refers to the amount of compound ( ⁇ g/g kidney) found in the kidney at 4 or 16 hours after a 17.2 mg/kg sc dose in a mouse.
• WO 2015/135976 polymyxin nonapeptides with an N-terminal ⁇ -aminopropyl group substituted with a phenyl or benzyl moiety.
• the phenyl or benzyl substituent is provided at the ⁇ -position, rather than ⁇ -position, relative to the —X— group.
• WO 2015/135976 also describes a polymyxin nonapeptide with an N-terminal ⁇ -aminoethyl group substituted at the ⁇ -position with a benzyl group.
• the amino functionality is provided at the ⁇ -position rather than the ⁇ -position, relative to the —X— group.
• polymyxin nonapeptides with modified N-terminal groups particularly those including ⁇ -aryl or ⁇ -aralkyl groups.
• these known compounds have a lipophilic amino acid residue at position 6, such as a cyclohexylalanine residue, whilst the compounds of the present case have, for example, a phenylalanine, leucine, norleucine, valine or norvaline residue at position 6.
• These known compounds again are inferior to the compounds of the current invention in view of the poor combination of cytotoxicity and kidney drug levels after dosage.
• the compounds of formula (I) are N-terminal derivatives of the polymyxin nonapeptide series of compounds.
• the core of the compound of formula (I) is a derivative of a nonapeptide, such as the Polymyxin B nonapeptide (PMBN, Polymyxin B 2-10), where the amino acid residue at position 3 is substituted with Dap.
• the amino acid residues at positions 6 and/or position 7 are substituted with another amino acid, such as described herein.
• the compounds of formula (I) have a group —X—R 15 at the N terminal of the polymyxin core. This is described in detail below.
• the amino acid residue at position 6 may be the same as the amino acid residue at position 6 of Polymyxin B. That is, R 1 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached may be a D-phenylalanine residue.
• the amino acid residue at position 6 may the same as the amino acid residue at position 6 of Colistin. That is, R 1 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached may a D-leucine residue.
• —R 1 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached is a phenylalanine, leucine, norleucine, valine or norvaline residue.
• the amino acid residue may be the D-form.
• —R 1 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached is an amino acid residue, such as a phenylalanine, leucine or norleucine residue.
• the amino acid residue may be the D-form.
• —R 1 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached is a phenylalanine residue, for example a D-phenylalanine, or a leucine residue, such as a D-leucine residue.
• substitution of the amino acid residue at position 6 is known from, for example, WO 2016/083531.
• the group —R 2 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached, corresponds to the amino acid residue at position 7 in the polymyxin series of compounds.
• the group —R 2 is C 1-4 alkyl optionally substituted with one hydroxyl group.
• —R 2 is C 1-4 alkyl. This group is not substituted.
• —R 2 is C 3-4 alkyl, such as C 4 alkyl, optionally substituted with one hydroxyl group, such as unsubstituted.
• the group amino acid residue at position 7 may the same as the amino acid residue at position 7 of Polymyxin B and Colistin. That is, R 2 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached may a L-Leu residue.
• —R 2 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached is a leucine, iso-leucine, phenylalanine, threonine, valine, nor-valine, alanine, threonine or aminobutyrate residue.
• the amino acid residue may be the L-form.
• —R 2 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached is a leucine, aminobutyrate, or threonine residue.
• the amino acid residue may be the L-form.
• amino acid residue at position 7 may be substituted with another amino acid residue with respect to Polymyxin B and Colistin.
• —R 2 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached is a leucine, threonine or aminobutyric acid (Abu) residue, such as L-leucine, L-threonine or L-Abu.
• Abu aminobutyric acid
• substitution of the amino acid residue at position 7 is described in, for example, WO 2016/083531 and Velkov et al., amongst others.
• the group —R 3 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached, corresponds to the amino acid residue at position 10 in the polymyxin series of compounds.
• the group —R 4 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached, corresponds to the amino acid residue at position 3 in the polymyxin series of compounds.
• the amino group within the Dap side chain may be protected, for example the amino group may be protected with Boc.
• the group —R 8 together with the carbonyl group and nitrogen beta to the carbon to which it is attached via a hydroxymethylene spacer (—CH(OH)—), corresponds to the amino acid residue at position 2 in the polymyxin series of compounds.
• the amino acid residue at position 2 may the same as the amino acid residue at position 2 of Polymyxin B and Colistin. That is, R 1 together with the carbonyl group and nitrogen beta to the carbon to which it is attached via a hydroxymethylene spacer may be a L-Threonine residue.
• amino acid residue including the group —R 8 corresponds to position 2 in the polymyxin series of compounds.
• —R 8 is methyl.
• the resulting amino acid is therefore Thr.
• —R 8 is H.
• the resulting amino acid is therefore Ser.
• R 8 is methyl
• the group —X— is —C(O)—*.
• the asterisk indicates the point of attachment to NH, the amino terminal of the polymyxin nonapeptide core, such as the amino acid at position 2.
• the left-hand side of the group —X— is the point of attachment to —R 15 .
• the compounds of the invention contain a stereocentre at the ⁇ -position of the ⁇ -aminopropyl group in the N-terminal moiety, —R 15 . It has been found that one of the stereoisomers is associated with lower cytotoxicity and lower kidney drug levels. This stereoisomer is the stereoisomer that elutes more rapidly on reverse phase chromatography, as described in detail in the worked examples of the present case.
• R 15 may be:
• This stereoform is the slower eluting stereoform.
• the group —R 15 is:
• the groups —R 16 and —R 17 are both hydrogen.
• the group may be a covalent bond methylene (—CH 2 —).
• -L- is a covalent bond
• the group —Ar is an aryl group, such as a carboaryl or heteroaryl group.
• the aryl group is optionally substituted.
• the aryl group may be a C 6 aryl group, such as phenyl, or a C 5 aryl group, such as thiophene.
• the group —Ar may be phenyl, and this is optionally substituted, such as substituted. In one embodiment, —Ar is unsubstituted phenyl.
• the aryl group may be substituted, such as with one or more groups —R S .
• Each group —R S is independently selected from halo, alkyl, haloalkyl and aryl, such as halo and alkyl.
• An aryl group may be substituted with one, two or three —R S groups, such as one or two groups, such as one group (monosubstituted).
• a halo group may be selected from fluoro, chloro, bromo and iodo, and may be selected from fluoro and chloro, such as chloro.
• a halo group may be chloro
• An alkyl group may be C 1-6 alkyl, such as C 1-4 alkyl, such as C 1-3 alkyl, such as C 3 alkyl.
• An alkyl group may be selected from methyl, ethyl, and propyl, including n-propyl and i-propyl.
• An alkyl group may be i-propyl.
• a haloalkyl group may be a C 1-6 alkyl group, such as C 1-4 alkyl, such as C 1-2 alkyl, substituted with one or more halo groups.
• the alkyl group may be per-substituted with halo, such as per-substituted with fluoro.
• a haloalkyl group may be trifluoromethyl.
• the aryl group may itself be optionally substituted with one, two or three —R Ar groups, such as one or two groups, such as one group (monosubstituted).
• R Ar is independently selected from halo, alkyl, haloalkyl and aryl, such as halo and alkyl. These groups may have the same meanings as the groups described above, except that the aryl group is not substituted.
• the group —Ar may be halophenyl or alkylphenyl.
• the alkylphenyl may be selected from 2-alkylphenyl, such as 2-isopropylphenyl, 3-alkylphenyl, such as 3-isopropylphenyl, and 4-alkylphenyl, such as 4-isopropylphenyl
• the group —Ar may be 2-chlorophenyl or 3-isopropylphenyl.
• the group —Ar may be 2-chlorophenyl.
• the group —R 15 may be selected from the groups below:
• the group —R 15 may be selected from the groups below:
• the group —R 15 may be selected from the groups below:
• the compound of formula (I) is a compound of formula (la) having the orientation of R 1 , R 2 , R 3 , and R 4 moieties as depicted below:
• —R 1 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached, is a D-phenylalanine, D-leucine or D-norleucine.
• —R 2 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached, is a L-leucine, L-aminobutyrate, or L-threonine residue.
• the compounds of formula (Ia) may be compounds where -L- is a covalent bond or —CH 2 —, and —Ar is phenyl optionally substituted with one or two groups selected from the group consisting of halo, C1-C6 alkyl, optionally substituted aryl and optionally substituted heteroaryl.
• —Ar may be phenyl optionally substituted with one or two groups selected from the group consisting of chloro, bromo, thiophenyl, phenyl, methyl, isopropyl, and isobutyl.
• the compound of formula (I) may be a compound of formula (IIb) as shown below:
• the compounds for use in the present case are based on modified forms of known polymyxin compounds, such as Polymyxin B nonapeptide and Colistin nonapeptide
• Polymyxin B nonapeptide has the structure shown below:
• the compounds of the invention are derivatives of polymyxin B nonapeptide, where (i) the N terminal amino group, —NH 2 , is replaced with the group —NH—X—R 15 as described herein; (ii) the amino acid residue at position 3 is substituted with Dap, and optionally (iii) the amino acid residues at 2, 6 and/or 7 are substituted with another amino acid residue.
• the compounds of the invention are represented by the formula (I) where the amino acids at positions 2, 3, 6, 7 or 10 are determined by the nature of the groups R 8 , R 4 , R 1 , R 2 and R 3 respectively.
• Compounds of the invention, which include the variants described above, are biologically active.
• the preparation of the compounds of the invention will be familiar to those of skill in the art, particularly having knowledge of the methods described in WO 2015/135976 for the preparation of modified polymyxin nonapeptides.
• the methods described in the art may be readily adapted for use in the preparation of the compounds of the present case, taking into account the novel N terminal groups employed in the present case.
• a compound of the invention may be prepared by coupling a suitably protected polymyxin nonapeptide intermediate with a carboxylic acid having the group —R 15 .
• the product of this reaction is typically the protected form of the compound of formula (I). Removal of the protecting groups may be undertaken as desired. This is the general strategy known from WO 2015/135976.
• a suitably protected nonapeptide intermediate may itself be prepared according to the methods set out in in WO 2015/135976. As described herein, a suitably protected nonapeptide intermediate may also be prepared by solid phase synthesis of a linear nonapeptide, followed by cleavage of the linear form from the solid support and then subsequent cyclisation of that linear form between the amino residues at positions 4 and 10.
• Compounds of the invention may be made by conventional peptide synthesis, using methods known to those skilled in the art. Suitable methods include solid-phase synthesis such as described by de Visser et al, J. Peptide Res, 61, 2003, 298-306, Vaara et al, Antimicrob. Agents and Chemotherapy, 52, 2008. 3229-3236, or by Velkov et al. ACS Chem. Biol. 9, 2014, 1172. These methods include a suitable protection strategy, and methods for the cyclisation step.
• the compound of formula (I) may be at least partially purified, for example to separate diastereomeric forms of the product.
• the compound of formula (X) finds use in the preparation of the compounds of formula (I).
• the compound (X) such as in its protected form, is coupled with a protected polymyxin nonapetide of formula (XI) to yield a protected form of the compound of formula
• the amino functionality which is the group —NR 16 R 17 , is protected.
• the amino functionality in the compound of formula (X) is Boc- or CBZ-protected.
• —R 16 is hydrogen and —R 17 is —C(O)O-t-Bu or —C(O)O—Bn.
• the compound of formula (XI) is a compound of formula (I) except that the group R 15 —X— is hydrogen, and salts, solvates, and protected forms thereof.
• the carboxylic acid compound of formula (X) may be coupled with the amino compound of formula (XI) using conventional amide coupling conditions.
• the compound of formula (X) may be used in activated form, which form may be generated in situ, for reaction with the compound of formula (XI).
• the activated form may be generated in situ through the appropriate choice of coupling agents in the amide bond-forming reaction, optionally in the presence of base.
• the carboxylic acid of formula (X) may be activated by reaction of a standard activating agent, such as a carbodiimide, such as DIC (N,N′-diisopropylcarbodiimide) or EDCI (water soluble carbodiimide; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide).
• a standard activating agent such as a carbodiimide, such as DIC (N,N′-diisopropylcarbodiimide) or EDCI (water soluble carbodiimide; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide).
• DIC N,N′-diisopropylcarbodiimide
• EDCI water soluble carbodiimide; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
• the carboxylic acid may be activated by a hydroxybenzotriazole or a hydroxyazabenzotriazole, such as HOBt (1-hydroxy-benzotriazole) or HOAt (1-hydroxy-7-aza-benzotriazole).
• the activated form of the acid is an ester.
• the ester may be formed via the carbodiimide-activated form, or it may be formed from the carboxylic acid directly, for example using an appropriate reagent, such as HATU, HBTU, PyBOP, PyBROP, or TBTU.
• an appropriate reagent such as HATU, HBTU, PyBOP, PyBROP, or TBTU.
• An organic base may be present for the formation of the activated form, such as DIPEA or TEA.
• Compounds of the invention may be provided in a protected form.
• reactive functionality such as amino functionality
• a protecting group is provided to mask the reactive functionality, and this protecting groups may be removed at a later stage of the synthesis to reveal the original reactive functionality.
• the protected form is a compound where amino, hydroxyl, thiol, and/or carboxyl functionality is protected (masked) by a protecting group. In one embodiment, the protected form is a compound where the side chain functionality of the amino acids residues with the compound are protected.
• the amino acid residues at positions 5, 8 and 9 are Dab residues, and the side chain of the Dab residue includes amino functionality.
• the amino acid functionality of each Dab residue may be protected with an amino protecting group, as described herein.
• the amino acid residue at position 3 is Dap, and the side chain of this amino acid residue includes amino functionality.
• the group —R 15 contains amino functionality in the form of the group —R 16 R 17 , for example where each of —R 16 and —R 17 is hydrogen.
• the amino functionality may be protected with amino protecting groups, as described herein.
• a protected polymyxin compound may be prepared from appropriately protected amino acid starting materials.
• Velkov et al. describe the step-wise preparation of polymyxin compounds on the solid-phase using suitably protected amino acid.
• the use of protected-forms of threonine and Dab is disclosed (see Supplementary Information).
• a protecting group is it is removable under conditions that do not substantially disrupt the structure of the polymyxin core, for example conditions that do not alter the stereochemistry of the amino acid residues.
• the protecting groups are acid-labile, base labile, or are removable under reducing conditions.
• Example protecting groups for amino functionality include Boc (tert-butoxycabonyl), Bn (benzyl, Bzl), CbZ (benzyloxycarbonyl, Z), 2-CL-Z (2-chloro), ivDde (1-[4,4-dimethyl-2,6-dioxocylcohex-1-ylidene]-3-methylbutyl), Fmoc (fluorenylmethyloxycarbonyl), HSO 3 -Fmoc (sulfonylated Fmoc, such as 2-sulfo-Fmoc, as described in e.g. Schechter et al, J.
• Example protecting groups for aromatic nitrogen functionality includes Boc, Mtt, Trt and Dnp (dinitrophenyl).
• the protecting group for amino functionality is selected from Boc, ivDde, CbZ, Bn and Fmoc and HSO 3 -Fmoc.
• the protecting group for amino functionality is Boc, ivDde, Fmoc or CbZ, such as Boc, ivDde or Cbz.
• Boc protection may be provided for the amino functionality present in the side chains of the amino acid residues present at positions 5, 8 and 9, and optionally position 3.
• Example protecting groups for hydroxyl functionality include Trt (trityl), Bn (benzyl), and tBu (tert-butyl).
• the protecting group for hydroxyl functionality is tBu.
• protecting groups include silyl ether protecting groups, such as TMS, TES, TBS, TIPS, TBDMS, and TBDPS. Such protecting groups are removable with TBAF, for example.
• Example protecting groups for carboxyl functionality include Bn (benzyl, Bz), tBu (tert-butyl), TMSET (trimethylsilylethyl) and Dmab ( ⁇ 1-[4,4-dimethyl-2,6-dioxocylcohex-1-ylidene]-3-methylbutyl ⁇ amino benzyl).
• Example protecting groups for aromatic nitrogen functionality for example where such functionality is present in the group —Ar, includes Boc, Mtt, Trt and Dnp (dinitrophenyl).
• only some types of functionality are protected.
• only amino groups may be protected, such as amino groups in the side chain of an amino acid residue.
• amino groups and hydroxyl groups are protected.
• a compound of the invention such as a compound of formula (I), (II) or (III), may have a partition-coefficient, such as expressed as a LogP value, within certain limits.
• the partition-coefficient may provide an indication of the lipophilicity of the compound.
• the inventors have established that compounds having a higher lipophilicity have poorer cytotoxicity.
• the compounds of the invention typically have LogP values that are associated with lower cytotoxicity, such as the LogP values described below.
• a LogP value for a compound may be determined experimentally (for example by partition of the compound between octanol and water), or it may be predicted using standard computational methods.
• a reference to LogP may be a reference to ALogP, which may be determined using the methods described by Ghose et al. J. Phys. Chem. A, 1998, 102, 3762-3772, the contents of which are hereby incorporated by reference in their entirety.
• ALogP is the Ghose/Crippen group-contribution estimate for LogP.
• a compound has a LogP value, such as an ALogP, value, of at most ⁇ 6.4, at most ⁇ 6.3, at most ⁇ 6.2, at most ⁇ 6.1, at most ⁇ 6.0, at most ⁇ 5.9, or at most ⁇ 5.8.
• LogP value such as an ALogP
• a compound has a LogP value within a range having upper and lower limits appropriately selected from the limits given above, for example within the range ⁇ 5.8 to ⁇ 8.0, such as ⁇ 6.0 to ⁇ 6.7, such as ⁇ 6.3 to ⁇ 6.7. These ranges may be selected when the group —R 2 is unsubstituted alkyl.
• the compound has a LogP value within the range ⁇ 6.7 to ⁇ 7.4. This range may be selected when the group —R 2 is alkyl substituted with one hydroxyl group.
• a compound having optimal LogP values may be obtained by selecting the —R 15 group of the present case, together with appropriate choices of amino acid residues at position 6 and/or 7 (such as with appropriate selection of —R 1 and/or —R 2 ).
• the compounds of formula (I), (II) or (III) may each be used together with a second active agent.
• the inventors have found that such combinations have greater biological activity than would be expected from the individual activity of both compounds.
• the compounds of formula (I), (II) or (III) can be used to potentiate the activity of the second active agent.
• the compounds of formula (I), (II) or (III) may be used together with a second active agent to enhance the antimicrobial activity of that agent, for example against Gram-negative bacteria.
• the second active agent is an agent having a measured MIC value against a particular microorganism, such as a bacterium, that is less than 10, less than 5, or less than 1 micrograms/mL.
• the microorganism may be a Gram-negative bacteria, such as a Gram-negative bacteria selected from the group consisting of E. coli, S. enterica, K. pneumoniae, K. oxytoca; E. cloacae, E. aerogenes, E. agglomerans, A. calcoaceticus, A. baumannii; Pseudomonas aeruginosa , and Stenotrophomonas maltophila.
• the second active agent is an agent having a measured MIC value against a particular microorganism, such as a Gram-negative bacterium, that is more than 4, more than 8, more than 16 or more than 32 micrograms/mL.
• the second active agent may be active against Gram-positive bacteria.
• the second active agent is an agent having a measured MIC value against a particular Gram-positive bacterium that is less than 10, less than 5, or less than 1 micrograms/mL.
• the compound of formula (I), (II) or (III) acts to facilitate the uptake of the second active agent into the Gram-negative bacterial cell.
• the second active agent is therefore able to act on a target within the Gram-negative bacterial cell, which target may be the same as the second active agent's target in a Gram-positive bacterial cell.
• the Gram-positive bacteria may be selected from the group consisting of Staphylococcus and Streptococcus bacteria, such as S. aureus (including MRSA), S. epidermis, E. faecalis , and E. faecium.
• second active agents that have activity against Gram-positive bacteria (at the MIC values given above, for example), and moderate activity against Gram-negative bacteria, include rifampicin, novobiocin, macrolides, pleuromutilins.
• a compound having moderate activity against Gram-negative bacteria may have a measured MIC value against a Gram-negative bacterium that is less than 32, less than 64, or less than 128 micrograms/mL.
• agents having activity against Gram-positive bacteria and which are essentially inactive against Gram-negative bacteria include fusidic acid, oxazolidinines (e.g. linezolid), glycopeptides (e.g. vancomycin), daptomycin and lantibiotics.
• a compound having essentially no activity against Gram-negative bacteria may have a measured MIC value against a Gram-negative bacterium that is more than 32, more then 64, more than 128, more than 256 micrograms/mL.
• the active agent may be selected from the group consisting of rifampicin (rifampin), rifabutin, rifalazil, rifapentine, rifaximin, aztreonam, oxacillin, novobiocin, fusidic acid, azithromycin, ciprofloxacin, meropenem, tigecycline, minocycline, erythromycin, clarithromycin and mupirocin, and pharmaceutically acceptable salts, solvates and prodrug forms thereof.
• rifampicin rifampin
• rifabutin rifalazil
• rifapentine rifaximin
• aztreonam oxacillin
• fusidic acid azithromycin
• ciprofloxacin ciprofloxacin
• meropenem meropenem
• tigecycline minocycline
• erythromycin clarithromycin and
• the present inventors have found that the polymyxin compounds of formula (I), (II) or (III) may be used together with certain compounds in the rifamycin family to treat microbial infections.
• the rifamycin family includes isolates rifamycin A, B, C, D, E, S and SV, and synthetically derivatised versions of these compounds, such as rifampicin (rifampin), rifabutin, rifalazil, rifapentine, and rifaximin, and pharmaceutically acceptable salts and solvates thereof.
• the active agent is rifampicin (rifampin) and pharmaceutically acceptable salts, solvates and prodrug forms thereof.
• salts of compound of formula (I), (II) and (III) include all pharmaceutically acceptable salts, such as, without limitation, acid addition salts of strong mineral acids such as HCl and HBr salts and addition salts of strong organic acids such as a methanesulfonic acid salt. Further examples of salts include sulfates and acetates such as acetate itself, trifluoroacetate or trichloroacetate.
• the compounds of the present disclosure are provided as a sulfate salt or a trifluoroacetic acid (TFA) salt. In one embodiment the compounds of the present disclosure are provided as acetate salts, such as acetate.
• a compound of formula (I), (II) or (III) can also be formulated as a prodrug.
• Prodrugs can include an antibacterial compound herein described in which one or more amino groups are protected with a group which can be cleaved in vivo, to liberate the biologically active compound.
• the prodrug is an “amine prodrug”.
• examples of amine prodrugs include sulphomethyl, as described in e.g., Bergen et al, Antimicrob. Agents and Chemotherapy, 2006, 50, 1953 or HSO 3 -FMOC, as described in e.g. Schechter et al, J. Med Chem 2002, 45(19) 4264, and salts thereof. Further examples of amine prodrugs are given by Krise and Oliyai in Biotechnology: Pharmaceutical Aspects, 2007, 5(2), 101-131.
• a compound of formula (I), (II) or (III) is provided as a prodrug.
• a reference to a compound of formula (I), (II) or (III), or any other compound described herein, is also a reference to a solvate of that compound.
• solvates include hydrates.
• a compound of formula (I), (II) or (III), or any other compound described herein includes a compound where an atom is replaced by a naturally occurring or non-naturally occurring isotope.
• the isotope is a stable isotope.
• a compound described here includes, for example deuterium containing compounds and the like.
• H may be in any isotopic form, including 1 H, 2 H (D), and 3 H (T);
• C may be in any isotopic form, including 12 C, 13 C, and 14 C;
• O may be in any isotopic form, including 18 O and 18 O; and the like.
• Certain compounds of formula (I), (II) or (III), or any other compound described herein may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and I-forms; (+) and ( ⁇ ) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; ⁇ - and ⁇ -forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers
• isomers are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space).
• a reference to a methoxy group, —OCH 3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH 2 OH.
• a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl.
• a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C 1-6 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
• a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof.
• Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
• One aspect of the present invention pertains to compounds in substantially purified form and/or in a form substantially free from contaminants.
• the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.
• the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form.
• the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds.
• the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer.
• the substantially purified form refers to a mixture of enantiomers.
• the substantially purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate).
• the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.
• the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight.
• the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.
• the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure.
• 60% optically pure i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer
• at least 70% optically pure e.g., at least 80% optically pure, e.g., at least 90% optically pure, e
• the compounds of formula (I), (II) or (III), or pharmaceutical formulations containing these compounds are suitable for use in methods of treatment and prophylaxis.
• the compounds may be administered to a subject in need thereof.
• the compounds are suitable for use together with an active agent (“a second active agent”), for example a second active agent that is an antimicrobial agent.
• the compounds of formula (I), (II) or (III) are for use in a method of treatment of the human or animal body by therapy.
• a compound of formula (I), (II) or (III) may be administered to a mammalian subject, such as a human, in order to treat a microbial infection.
• Another aspect of the present invention pertains to use of a compound of formula (I) or (II) in the manufacture of a medicament for use in treatment.
• the medicament comprises a compound of formula (I), (II) or (III).
• the medicament is for use in the treatment of a microbial infection.
• microbial infection refers to the invasion of the host animal by pathogenic microbes. This includes the excessive growth of microbes that are normally present in or on the body of an animal. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host animal. Thus, an animal is “suffering” from a microbial infection when excessive numbers of a microbial population are present in or on an animal's body, or when the presence of a microbial population(s) is damaging the cells or other tissue of an animal.
• the compounds may be used to treat a subject having a microbial infection, or at risk of infection from a microorganism, such as a bacterium.
• the microbial infection may be a bacterial infection such as a Gram-negative bacterial infection.
• Gram-negative bacteria examples include, but are not limited to, Escherichia spp., Klebsiella spp., Enterobacter spp., Salmonella spp., Citrobacter spp., and other Enterobacteriaceae, Pseudomonas spp., Acinetobacter spp., Stenotrophomonas , and Legionella and numerous others.
• Medically relevant Gram-negative bacilli include a multitude of species. Some of them primarily cause respiratory problems ( Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa ), primarily urinary problems ( Escherichia coli, Enterobacter cloacae ), and primarily gastrointestinal problems ( Salmonella enterica ).
• Gram-negative bacteria associated with nosocomial infections include Acinetobacter baumannii , which causes bacteraemia, secondary meningitis, and ventilator-associated pneumonia in intensive-care units of hospital establishments.
• the Gram-negative bacterial species is selected from the group consisting of E. coli, S. enterica, K. pneumoniae, K. oxytoca; E. cloacae, E. aerogenes, E. agglomerans, A. calcoaceticus, A. baumannii; Pseudomonas aeruginosa , and Stenotrophomonas maltophila.
• the Gram-negative bacterial species is selected from the group consisting of E. coli, K. pneumoniae, Pseudomonas aeruginosa , and A. baumannii.
• the compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of skin and soft tissue infections, gastrointestinal infection, urinary tract infection, pneumonia, sepsis, intra-abdominal infection and obstetrical/gynaecological infections.
• the infections may be Gram-negative bacterial infections.
• the compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of Pseudomonas infections including P. aeruginosa infection, for example skin and soft tissue infections, gastrointestinal infection, urinary tract infection, pneumonia and sepsis.
• Pseudomonas infections including P. aeruginosa infection, for example skin and soft tissue infections, gastrointestinal infection, urinary tract infection, pneumonia and sepsis.
• the compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of Acinetobacter infections including A. baumanii infection, for pneumonia, wound infections, urinary tract infection and sepsis.
• the compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of Klebsiella infections including K. pneumoniae infection, for pneumonia, intra-abdominal infection, urinary tract infection, meningitis and sepsis.
• the compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of E. coli infection including E. coli infections, for bacteraemia, cholecystitis, cholangitis, intra-abdominal infection, urinary tract infection, neonatal meningitis and pneumonia.
• compositions comprising the same may be used together with an active agent in methods of treatment.
• the active agent may be an agent that has activity against the microorganism.
• the active agent may be active against Gram-negative bacteria.
• the active agent may be active against a microorganism selected from the list given above.
• the second active agent has an MIC value of 10 micrograms/mL or less against a microorganism such as E. coli , in the absence of the compound of formula (I), (II) or (III).
• the microorganism may be a microorganism selected from the group above.
• the compounds of formula (I) are suitable for use in the treatment of fungal infections, for example in combination together with an antifungal agent.
• the antifungal agent may be selected from a polyene antifungal, for example amphotericin B, an imidazole, triazole, or thiazole antifungal, for example miconazole, fluconazole or abafungin, an allylamine, an echinocandin, or another agent, for example ciclopirox.
• treatment pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition.
• Treatment as a prophylactic measure i.e., prophylaxis
• treatment is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term “treatment.”
• terapéuticaally-effective amount pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
• treatment includes combination treatments and therapies, as described herein, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
• a compound of formula (I), (II) or (III) may be administered in conjunction with an active agent. Administration may be simultaneous, separate or sequential.
• a compound of formula (I), (II) or (III) and a second active agent are administered to a subject in a single dose by the same route of administration.
• a compound of formula (I), (II) or (III) and a second active agent are administered to a subject by two different routes of administration which occur at the same time. This may occur for example where one agent is administered by infusion and the other is given orally during the course of the infusion.
• quential it is meant that the two agents are administered at different points in time, provided that the activity of the first administered agent is present and ongoing in the subject at the time the second active agent is administered.
• a sequential dose will occur such that the second of the two agents is administered within 48 hours, such as within 24 hours, such as within 12, 6, 4, 2 or 1 hour(s) of the first agent.
• the active agent may be administered first, followed by the compound of formula (I), (II) or (III).
• the order and timing of the administration of the compound and second active agent in the combination treatment will depend upon the pharmacokinetic properties of each.
• the amount of the compound of formula (I), (II) or (III) to be administered to a subject will ultimately depend upon the nature of the subject and the disease to be treated. Likewise, the amount of the active agent to be administered to a subject will ultimately depend upon the nature of the subject and the disease to be treated.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound of formula (I), (II) or (III) together with a pharmaceutically acceptable carrier.
• the pharmaceutical composition may additionally comprise a second active agent.
• the second active agent may be separately formulated from the compound of formula (I), (II) or (III). The comments below made in relation to the compound of formula (I), (II) or (III) may therefore also apply to the second active agent, as separately formulated.
• compositions, preparation, medicament comprising at least one compound of formula (I), (II) or (III), as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
• the formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
• the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition
• a pharmaceutical composition comprising admixing at least one compound of formula (I), (II) or (III), as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.
• the composition optionally further comprises the second active agent in a predetermined amount.
• pharmaceutically acceptable pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
• Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.
• the formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound of formula (I) or (II) with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
• carriers e.g., liquid carriers, finely divided solid carrier, etc.
• the formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
• Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
• solutions e.g., aqueous, non-aqueous
• suspensions e.g., aqueous, non-aqueous
• Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.
• the compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients.
• the compound may be presented in a liposome or other microparticulate which is designed to target the compound, for example, to blood components or one or more organs. Where a liposome is used, it is noted that the liposome may contain both the compound of formula (I), (II), (III) and a second active agent.
• Formulations suitable for oral administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.
• Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
• Losenges typically comprise the compound in a flavoured basis, usually sucrose and acacia or tragacanth.
• Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia.
• Mouthwashes typically comprise the compound in a suitable liquid carrier.
• Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.
• Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
• solutions e.g., aqueous, non-aqueous
• suspensions e.g., aqueous, non-aqueous
• emulsions e.g., oil-in-water, water-in-oil
• mouthwashes e.g., losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
• Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
• solutions e.g., aqueous, non-aqueous
• suspensions e.g., aqueous, non-aqueous
• emulsions e.g., oil-in-water, water-in-oil
• suppositories e.g., pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
• Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
• Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl
• Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
• the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.
• Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach.
• Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.
• Creams are typically prepared from the compound and an oil-in-water cream base.
• the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
• the topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
• Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
• an emulsifier otherwise known as an emulgent
• a hydrophilic emulsifier may be included together with a lipophilic emulsifier which acts as a stabiliser. It is also possible to include both an oil and a fat.
• the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax
• the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
• Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
• Formulations suitable for intranasal administration, where the carrier is a liquid include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound.
• a dry powder delivery may be used as an alternative to nebulised aerosols.
• Formulations suitable for intranasal administration, where the carrier is a solid include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
• Formulations suitable for pulmonary administration include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, carbon dioxide, or other suitable gases.
• a formulation for pulmonary administration may be formulated for administration from a nebuliser or a dry powder inhaler.
• the formulation may be provided with carriers or liposomes to provide a suitable particle size to reach the appropriate parts of the lung, to aid delivery of an appropriate does to enhance retention in the lung tissue.
• Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.
• Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
• a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
• Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.
• Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate).
• sterile liquids e.g., solutions, suspensions
• Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
• excipients include, for example, water, alcohols, sugars, polyols, glycerol, vegetable oils, and the like.
• suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
• the concentration of the compound in the liquid is from about 1 ng/mL to about 500 ⁇ g/mL, for example about 1 ng/mL to about 100 ⁇ g/mL, for example from about 10 ng/mL to about 10 ⁇ g/mL, for example from about 10 ng/mL to about 1 ⁇ g/mL.
• the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
• sterile liquid carrier for example water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
• the methods of the invention may comprise administering to a subject an effective amount of a compound of formula (I), (II) or (III) so as to provide an antimicrobial effect.
• the compound of formula (I), (II) or (III) may be administered at an amount sufficient to potentiate the activity of a second active agent.
• the second active agent is administered to a subject at an effective amount so as to provide an antimicrobial effect.
• the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound of formula (I), (II) or (III) or the active agent, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient.
• the amount of compound of formula (I), (II) or (III) or the active agent and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
• Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
• a suitable dose of a compound of formula (I), (II) or (III) or the active agent is in the range of about 10 ⁇ g to about 250 mg (more typically about 100 ⁇ g to about 25 mg) per kilogram body weight of the subject per day.
• the compound of formula (I), (II) or (III) or the active agent is a salt, an ester, an amide, a prodrug, or the like
• the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.
• kits comprising (a) a compound of formula (I), (II) or (III), or a composition comprising a compound as defined in any one of formula (I), (II) or (III), e.g., typically provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on how to administer the compound or composition.
• the written instructions may also include a list of indications for which the compound of formula (I), (II) or (III) is a suitable treatment.
• the kit further comprises (c) a second active agent, or a composition comprising the second active agent.
• the written instructions may also include a list of indications for which the second active agent, together with the compound of formula (I), (II) or (III), is suitable for treatment.
• a compound of formula (I), (II) or (III), a second active agent, or a pharmaceutical composition comprising the compound of formula (I), (II) or (III), or the second active agent may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).
• Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection or infusion, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, sub
• the subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g
• the subject/patient is a human.
• a non-human mammal may be a rodent.
• Rodents include rats, mice, guinea pigs, chinchillas and other similarly-sized small rodents used in laboratory research.
• the crystal was kept at 302.71 K during data collection.
• Olex2 Dolomanov et al.
• the structure was solved with the SheIXT [2] structure solution program (Sheldrick A71) using Intrinsic Phasing and refined with the SheIXL [3] refinement package using Least Squares minimisation (Sheldrick C71).
• the compound was prepared from 2-chlorocinnamic acid following the methodology of 4-((tert-butoxycarbonyl)amino)-3-(3-chlorophenyl)butanoic acid as described in steps (i) to (iii) above.
• the compound was obtained as a colourless oil.
• Methyl 3-benzyl-4-nitrobutanoate ( Tetrahedron Asymmetry, 19, 2008, 945) was converted to the title compound following the methodology of 4-((tert-butoxycarbonyl)amino)-3-(3-chlorophenyl)butanoic acid as described in steps (i) to (iii) above.
• step (iii) The ethyl ester was hydrolysed with lithium hydroxide as previously described in step (iii) for the preparation of 4-((tert-butoxycarbonyl)amino)-3-(3-isobutylphenyl)butanoic acid, followed by chromatography on silica eluting with petroleum ether 40-60 and ethyl acetate (0-100%) to afford the title compound as a white solid (60%).
• 3,5-Dichlorobenzaldehyde was converted to the title compound using the methodology described above for the preparation of 4-((tert-butoxycarbonyl)amino)-3-(3-isobutylphenyl)butanoic acid in steps (i) through (iii).
• the ethyl ester was hydrolysed as described for step (v) in the preparation of (3-isobutylphenyl)butanoic acid, to afford the title compound as a colourless oil.
• Resin-bound peptide (3.93 g, corresponding to 1.5 mmol) was placed in a 500 mL conical flask and treated with 4% hydrazine in DMF (100 mL). The mixture was placed on a shaker and shaken gently for 30 mins. The mixture was poured into a sintered column, then washed with DMF (3 ⁇ 100 mL). Compressed air was applied to remove last traces of DMF. The procedure was then repeated with THF and with DCM.
• a linear peptide with orthogonal protection of the ⁇ -amino group of the Dab residue involved in cyclisation was constructed on-resin, with the C-terminal amino acid (typically Thr) attached to the solid phase.
• the resulting linear peptides were cyclised off-resin. Two general methods were used, as described below.
• Synthesis of the protected linear peptide was carried out on an automated peptide synthesizer using standard Fmoc solid phase peptide chemistry. Specifically, synthesis was undertaken using Fmoc-Thr(tBu)-PEG-PS resin as starting material. Coupling of the Fmoc-amino acids with CBZ protection on the terminal amino groups was performed using 5 molar equivalents (relative to resin loading) of Fmoc amino acid and HATU in DMF with activation in situ, using 10 molar equivalents of DIPEA. Fmoc deprotection was performed using 20% piperidine in dimethylformamide. BOC was used as the orthogonal protecting group on the Dab involved in cyclisation.
• the resin-bound linear peptide was treated with TFA/TIS/H 2 O (96/2/2v/v) for 2 h. to reveal the Dab residue involved in cyclisation, and to cleave the peptide from the resin.
• This material was cyclised using PyBop/HOBt/NMM (4/4/8 molar equivalents relative to the initial loading) in DMF for 3 h.
• the crude material was partially evaporated, taken up acetonitrile/water and lyophilised overnight.
• the CBZ groups were then removed using 10% Pd/C in Acetic acid/MeOH/water (5/4/1 v/v).
• Synthesis of the protected linear peptide was carried out on an automated peptide synthesizer using standard Fmoc solid phase peptide chemistry. Specifically, synthesis was undertaken using chlorotrityl chloride (CTC)-resin, pre-loaded with Fmoc-Thr(tBu)-OH (loading ⁇ 0.78 mmol/g), on 0.05-0.1 mmol scale. Coupling of the Fmoc-amino acids was performed using 5 molar equivalents (relative to resin loading) of Fmoc amino acid and HATU in DMF with activation in situ, using 10 molar equivalents of DIPEA. Fmoc deprotection was performed using 20% piperidine in dimethylformamide. The ivDde protecting group was used as orthogonal protection on the Dab residue involved in cyclisation.
• CTC chlorotrityl chloride
• Fmoc-Thr(tBu)-OH loading ⁇ 0.78 mmol/g
• the linear peptide was treated with 3% hydrazine in DMF (100 mL per 100 ⁇ mol, repeated twice) followed by washing with DMF ⁇ 3, EtOH ⁇ 3 and diethyl ether ⁇ 3.
• the partially deprotected linear peptide was then cleaved from the resin by washing the resin with 20% HFIP in DCM.
• the resulting residue was dissolved in 50% acetonitrile/water and freeze dried overnight.
• the protected linear peptide was dissolved in DMF (20 mL/mmol resin) cyclised with DPPA, (3 molar equivalents relative to the loading of the resin) and DIPEA (6 molar equivalents relative to the loading of the resin). This solution was stirred at room temperature overnight.
• the BOC groups were removed with TFA, and the crude peptide lyophilised.
• the crude product was purified and the diastereomers separated by preparative HPLC using the preparative HPLC conditions 4 described below. Note that the specific conditions were optimised for each pair of diastereomers.
• the enantiomerically pure amino acids were coupled to the N terminal of the nonapeptide compounds using the same conditions as described above in General method 3a for the enantiomerically mixed amino acids.
• Example (5) was assigned the (S) stereochemistry following X-ray determination of the absolute configuration of 4-amino-3-(3-chlorophenyl)butanoic acid derived from 4-((tert-butoxycarbonyl)amino)-3-(3-chlorophenyl)butanoic acid (isomer 2).
• Example 6 Coupling the 4-((tert-butoxycarbonyl)amino)-3-(3-chlorophenyl)butanoic acid (isomer 1, retention time 2.89 mins analytical method 1 or 2.796 mins analytical method 2) under the conditions of General Method 3a followed by deprotection afforded Example 6.
• Example (6) was assigned the (R) stereochemistry following X-ray determination of the absolute configuration of 4-amino-3-(3-chlorophenyl)butanoic acid derived from 4-((tert-butoxycarbonyl)amino)-3-(3-chlorophenyl)butanoic acid (isomer 1).
• AG1-X2 resin Bio-Rad Laboratories Ltd acetate form 200-4-mesh, was regenerated by washing with 10% aqueous acetic followed by 1% aqueous acetic acid, and placed in a fritted cartridge. A solution of the compound as a TFA salt in water was applied to the column, using a loading of 30 g resin to 1 g TFA salt, and the column allowed to drip under gravity, eluting with water. Product-containing fractions were combined and lyophilised to a white solid.
• Example 5 (faster isomer) 1 H NMR of the acetate salt (400 MHz, D 2 O): ⁇ (ppm) 0.70 (3 H, d, J 6.1 Hz), 0.77 (3 H, d, J 6.3 Hz), 0.78-0.90 (1H, m), 1.13 (3H, d, J 6.3 Hz), 1.17 (3H, d, J 6.4 Hz), 1.36-1.52 (2H, m), 1.75-2.06 (17 H, m, includes 1.91, s, OAc), 2.10-2.30 (4H, m), 2.72-2.91 (4H, m), 3.02-3.49 (14H, m), 4.12-4.32 (8 H, m), 4.48 (1 H, dd, J 5.6, 9.0 Hz), 4.54-4.60 (1H, m), 4.63-4.68 (1H, m), 7.25-7.41(9H, m). m/z 1145 [MH + ], 573 [M+2H] 2+
• Example 6 (slower isomer) 1 H NMR of the acetate salt (400 MHz, D 2 O): ⁇ (ppm) 0.60-0.67 (6 H, m), 0.69-0.84 (4 H, m), 1.16 (3H, d, J 6.4 Hz), 1.33-1.50 (2 H, m), 1.76-2.04 (19 H, m, includes 1.88, s, OAc), 2.06-2.26 (4 H, m), 2.67-2.86 (4 H, m), 3.00-3.46 (14 H, m), 3.98-4.04 (1 H, m) 4.14-4.30 (7H, m), 4.45 (1 H, dd, J 5.6, 9.0 Hz), 4.54 (1 H, appears as t, J 8.3 Hz), 4.72 (1 H, dd, J 5.0, 8.9 Hz), 7.20-7.40 (9 H, m). m/z 1145 [MH + ], 573 [M+2H] 2+ .
• Table 1 lists example compounds of the invention. These are compounds having the general structure shown below:
• the group R corresponds to —X—R 15 in the compounds of the invention, and this group is shown in the table together with the amino acid residues at positions 6 and 7 (AA-6 and AA-7 respectively, using the polymyxin numbering system), which correspond to the groups —R 1 and —R 2 respectively, when taken together with the carbonyl group and nitrogen alpha to the carbon to which they are attached.
• the group —R 3 taken together with the carbonyl group and nitrogen alpha to the carbon to which it is attached is L-Thr
• —R 4 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached
• L-Dap is —CH 2 NH 2
• —R 8 together with the carbonyl group and nitrogen alpha to the carbon to which it is attached, is L-Thr (thus —R 8 is methyl).
• Absolute stereochemistry in the sidechain -R has been assigned by comparison to Example 5 and Example 6 which have been correlated to material of known absolute stereochemistry.
• the table provides the HPLC retentions times for the example compounds.
• the HPLC conditions that were used for analysis are set out below.
• the inoculum was prepared by making a direct suspension of isolated colonies (selected from an 18-24 hour Mueller-Hinton agar plate) adjusted to the 0.5 McFarland standard. MIC testing was performed by two-fold serial antibiotic dilutions in cation-adjusted Mueller-Hinton Broth in sterile 96-well microtitre plates in a total volume of 170 ⁇ L (150 ⁇ L broth containing the antimicrobial agent, 20 ⁇ L inoculum). The assays were performed in duplicate. Plates were incubated aerobically without shaking for 18-20 hours at 35° C. with the MIC defined as the lowest concentration of drug that prevented visible growth. Several of the compounds were subjected to multiple tests, and where this is the case, the MIC presented is the median value obtained. The MIC values are quoted in ⁇ g/mL.
• the in vitro renal cell toxicity assay was performed according to the following protocol.
• HK-2 cells were maintained and assayed in Keratinocyte-SFM media supplemented with 5 ng/mL Epidermal Growth Factor (EGF) and 50 ⁇ g/mL Bovine Pituitary Extract (BPE). Cells were seeded at 7,500 cells per well in 96-well plates and allowed to adhere overnight. Polymyxin B (PMB) and test compounds were dissolved in 10% DMSO in water to give a stock solution of 20 and 60 mg/mL, respectively. The test compounds were diluted to give a top concentration of 3,000 or 1,000 ⁇ g/mL with semi-log dilutions to give a 9-point concentration range plus vehicle control.
• EGF Epidermal Growth Factor
• BPE Bovine Pituitary Extract
• PMB was also diluted to give a top concentration of 1,000 ⁇ g/ml with semi-log dilutions. Water and DMSO levels were kept constant at 5% and 0.5% respectively. The test compounds were incubated with cells for 24 h at 37° C. 5% CO 2 in a humidified atmosphere. CellTiter-Blue was diluted in PBS (1:4) and added 20% (v/v) and incubated at 37° C. for 2 h before fluorescent product was detected.
• IC 50 values are expressed relative to that for PMB in the same experiment. Where multiple determinations have been made, median values are presented.
• TFA Trifluoroacetic acid
• Kidney homogenates (100 ⁇ L) were mixed with methanol (190 ⁇ L) and TFA (110 ⁇ L, 10% v/v in water) and stored overnight at ⁇ 20° C. for protein precipitation. After 10 min. centrifugation at 13,000 rpm and 6° C., 200 ⁇ l of the supernatants were transferred into glass inserts and analysed by LC-MS-MS.
• Elevation of albumin, cystatin C or KIM-1 in the urine are signs of renal damage.
• Example 5 showed the lowest levels of all 3 biomarkers of renal toxicity.
• Reference Example D77 is described in WO 2015/135976.
• Reference Example 38 is described in WO 2016/083531.
• Example 5 or Example 9 groups None of the animals in the Example 5 or Example 9 groups showed any signs of degeneration or regeneration by histopathology. In contrast all 6 animals treated with PMB showed minimal tubular regeneration.
• Example 17 was compared with PMB.
• Example 5 The dose in these experiments was 5 ⁇ higher for Example 5 than PMB but signs of renal toxicity were reduced.
• Example 5 was more effective at 20 mg/kg and could also be dosed at higher levels to achieve a greater effect due to reduced toxicity.
• the compounds of the invention contain a stereocentre at the ⁇ -position of the ⁇ -aminopropyl group in the N-terminal moiety. Surprisingly it has been found that one of the stereoisomers at this position is usually associated with lower cytotoxicity and lower kidney drug levels. This is the stereoisomer that elutes more rapidly on reverse phase chromatography.
• the diastereomer which elutes faster from a reverse-phase HPLC column shows lower kidney exposure and lower cytotoxicity than the corresponding slower isomer.
• the faster diastereomer (Example 5) is derived from (S)-4-amino-3-(3-chlorophenyl butanoic acid, by small-molecule X-Ray analysis of the corresponding amino acid (as shown in the scheme below).
• the drug level refers to the amount of compound found in the kidney at 4 hours after 17.2 mg/kg sc dose ( ⁇ g/g) in a mouse model.
• PMB Polymyxin B
• Urine Biomarkers Dose Cystatin ⁇ -2 Compound (mg/kg) C microglobulin Kim-1 NGAL Albumin Vehicle — 22.24 0.05 17.01 0.00 3.44 PMB 12.5 29.45 3.43 21.89 0.30 3.75 PMB 25 44.51 43.83 3,446.42 4.37 7.73 Ex 24 25 31.04 0.05 27.61 0.00 4.78 Ex 24 50 33.97 13.22 171.59 0.63 6.95 Ex 24 75 81.44 68.51 1,203.41 3.71 10.93
• the urine biomarkers were normalized to urinary creatinine.
• Example 24 The renal histopathology at the 50 mg/kg dose of Example 24 was less severe than for PMB at a dose of 25 mg/kg, and the renal histopathology was similar at a 75 mg/kg dose of Example 24 compared with a dose of PMB at 25 mg/kg.

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